The aim of this application is to engineer neuro-vascular regenerative units ex vivo to provide optimized cellular therapies for disorders associated with progressive degeneration of the neurological and vascular systems, such as stroke. Our approach will produce systems that can grow human neural stem cells (NSC) ex vivo in a transplantable vascular microenvironment (or niche) that will enable the subsequent long-term survival, propagation and differentiation of NSC in vivo. Hence the overarching hypothesis of our proposed studies is that it will be possible to generate a neuro-vascular unit ex vivo that can be implanted into the CNS of stroke patients and provide a source of neuro-vascular cells that will continue to develop within the implant and integrate with existing tissue to prevent progressive loss and restore function. To begin testing this hypothesis, we have conducted immunohistochemical analyses, bioimaging and quantitative cytoarchitectural mapping of NSC niches including the sub-ventricular zone and olfactory bulb of the adult mouse brain and the sub-ventricular zone of the neonatal mouse to define the cellular architecture and physical interactions within these niches. We will similarly map the neurovascular architecture in the cortex, and determine how it is altered in response to stroke injury. We have also established in vitro co- culture systems and conducted studies to examine paracrine signaling between NSC and vascular endothelial cells isolated from these tissues, to determine how niche-specific flow forces impact such interactions, and how this affects the growth and differentiation of NSC. We have designed and generated biomaterials and bioreactors that will enable stem cell survival and propagation and fabrication of perfused microvascular networks. We have also established a rodent model of stroke-induced injury, devised methods to treat stroke with NSC, and developed MRI-assisted methods for tracking implanted cells and monitoring functional recovery of injured neural tissues. These proposed studies will therefore integrate knowledge and tools generated in our preliminary experiments. By recapitulating the NSC niche microenvironment ex vivo to fabricate transplantable neuro- vascular regenerative units, in subsequent phases of study we will be able to use these units to replace and repair stroke damaged tissue. These studies will represent only the initial application of this new approach for the correction of degenerative neural disorders. Some proposed studies will utilize NIH-approved human ES cell lines WA01 and WA09.